Physiological processes, which firstly ensure the fluidity of the blood in the vessel system and secondly make sure that extravascular blood losses are avoided by forming blood clots, are encompassed by the term hemostasis. A plurality of protein factors and also cellular components such as e.g. platelets (thrombocytes) are involved in regulating hemostasis. In the case of vessel damage, platelets firstly accumulate on the subendothelial collagen. This adhesion is mediated by adhesion proteins, such as the von Willebrand factor (VWF). During the adhesion process, the platelets are activated and release mediators from their granules, as a result of which the aggregation of further platelets and an increase in the activation are induced. This brings about a primary vessel-wall occlusion (primary hemostasis), which is only stabilized by further reactions of the plasmatic coagulation system (secondary hemostasis). Dysregulation of these processes can lead to thrombophilia or a tendency toward hemorrhage, and, depending on the degree of severity, can have life-threatening consequences.
Various in vitro test methods were developed in coagulation diagnostics and these can be used to determine whether the blood of a patient can coagulate properly or whether there is a coagulation defect. In the case of a coagulation defect, it is often necessary to obtain more precise information in respect of the cause of the present defect in order to be able to select the optimum therapeutic measures. An important sub-function of the coagulation system, which can be examined in targeted fashion, is primary hemostasis, which substantially depends on the function of the platelets.
Determining the function of the thrombocytes or platelets is a conventional object in hemostasis diagnostics and it is important in a multiplicity of clinical situations, e.g. in the early detection of cardiovascular diseases, for diagnosing hereditary or acquired platelet function defects, for ruling out bleeding complications prior to surgical interventions or for monitoring antithrombotic therapies. Medication that inhibits the aggregation of platelets is primarily used for prophylaxis and therapy of arterial thromboembolic events, such as a myocardial infarction or stroke. The most widespread drugs with platelet aggregation inhibiting effects are acetylsalicylic acid (ASA, Aspirin®) and the thienopyridines clopidogrel and ticlopidine.
The prior art has disclosed various methods for examining the platelet function. Determining the bleeding time is a global in vivo test that detects primary hemostasis. The bleeding time is determined by inflicting a small cut or piercing injury on the patient and measuring the time until the bleeding stops. This is a roughly informative test that is difficult to to standardize and mainly used in emergency situations in order to obtain an overview of primary hemostasis. Intake of platelet aggregation inhibitors leads to an increase in the bleeding time. A disadvantage of determining the bleeding time is that it is not possible to rule out a platelet function defect in the case of a normal bleeding time.
Various in vitro methods allow a significantly more sensitive detection of platelet function defects. In these methods, the aggregation of the platelets is usually induced in a whole-blood sample or a sample of platelet-rich plasma (PRP) by adding an activator and/or by applying shear forces and the aggregation reaction is measured. The most commonly used activators, which are used for inducing the platelet aggregation, are the following: ADP (adenosine 5′-diphosphate), collagen, epinephrine (adrenalin), ristocetin and various combinations thereof, and also thrombin, thrombin receptor activating protein (TRAP) or serotonin. In order to apply shear forces in vitro, which shear forces are an important trigger for platelet aggregation in vivo, different methods are used, such as e.g. stirring the platelet sample or guiding or pressing the platelet sample through cannulae or apertures with a small diameter.
In the case of conventional light-transmission aggregometry (LTA), which is also referred to as Born platelet aggregation, the aggregation efficiency of the platelets in the platelet-rich plasma is measured photometrically in an aggregometer in the presence of aggregation-inducing substances. As a result of aggregate formation, the light-transmission of the PRP sample is increased and so measuring the light-transmission makes it possible to determine e.g. the rate of the aggregate formation. Light-transmission aggregometry also makes it possible to detect therapeutic effects of platelet aggregation inhibitors, which are used medicinally. A disadvantage of light-transmission aggregometry is that only platelet-rich plasma can be used as a sample material. Platelet-rich plasma not only lacks important constituents of the blood, such as e.g. red and white blood cells, but also requires a time-consuming and error-prone sample preparation.
The VerifyNow® system (Accumetrics) is a development of light-transmission aggregometry, which allows the examination of the platelet function in whole-blood samples. In this system, the aggregation reaction of the platelets is increased by the addition of fibrinogen-coated microparticles.
An entirely different test principle for determining the platelet function is realized in the platelet function analyzer (PFA-100®, PFA-200 Siemens Healthcare Diagnostics). This is a universal, automated and standardized in vitro whole-blood test in which primary hemostasis is measured under flow conditions and hence in the presence of strong shear forces. In order to simulate the flow conditions and the shear forces, as are prevalent in relatively small arterial blood vessels, negative pressure of approximately −40 mbar is generated in a special measuring cell and the citrated whole blood, which is situated in a sample reservoir, flows through a capillary with a diameter of approximately 200 μm. The capillary opens into a measurement chamber closed off by a partition member, e.g. a membrane, which contains a capillary-like central opening (aperture) through which the blood passes owing to the negative pressure. One or more activators, which induce platelet aggregation, are usually added to the membrane, at least in the region around the aperture such that the blood flowing past this comes into contact with the aggregation-inducing substances in the region of the aperture. As a result of the induced adhesion and aggregation of the platelets, a platelet plug (blood clot) is formed in the region of the aperture and it closes the membrane opening and stops the blood flow. In this system, the time required to seal the membrane opening is measured. This so-called closure time correlates with the functional efficiency of the platelets. A measuring cell for use in a method for determining the platelet function on the basis of the closure time is described in e.g. WO 97/34698. By way of example, use is made of measuring cells equipped with a membrane coated with collagen (Col) and, additionally, with either ADP or epinephrine (Epi). Various partition members and the production and use thereof are described in e.g. WO 96/00899 A1.
Another test principle, in turn, for determining the platelet function is based on the forced passage of blood or platelet-rich plasma through a filter.
Uchiyama, S. et al. (Thrombosis Research 31: 99-116, 1983) describe the so-called filter bleeding time (FBT) test. In this method, whole blood at constant pressure (approximately 150 mmHg) is guided through a polyester fiber filter (Dacron®). Platelet aggregates plug the filter pores and reduce the flow rate. The bleeding time FBT is the time that passes between the start of the flow and the time at which the flow rate has dropped to below one drop per 30 seconds.
GB 2175691 A describes a development of the FBT test according to Uchiyama et al. Here a whole-blood sample is made to pass through a filter consisting of a fiber mesh by means of positive pressure. The filter has pores with different dimensions and allows the passage of particles with a diameter of up to 10 μm. The effect of this is that the sample can be pressed through the filter at lower pressures of only 20 to 100 mmHg. Relatively large platelet aggregates plug the pores and increasingly block the passage of sample material. Determining the flow rate or comparing the number of platelets in the filtered eluate with the number of platelets in the unfiltered sample provide an insight into the aggregation efficiency of the platelets and hence into the platelet function.
Another method for determining the platelet function, which is based on the principle of the forced passage of blood or platelet-rich plasma through a filter, is the so-called retention test Homburg (RTH) (Krischek, B. et al., Seminars in Thrombosis and Hemostasis 31(4): 449-457, 2005; Krischek, B. et al., Seminars in Thrombosis and Hemostasis 31(4): 458-463, 2005). In this method, whole blood or platelet-rich plasma is passed through a Porex® filter unit by means of a centrifugal force (10 minutes at 110×g), which filter unit has a height of 2.3 mm and a pore dimension of 16-22 μm. The difference between the number of platelets before and after the sample has passed through the filter is determined and the retention index (RI %) is calculated. A reduced retention of platelets in the filter indicates a loss of platelet function. An increased retention of platelets in the filter indicates an enhanced platelet activity.
A disadvantage of the two last-mentioned methods is that the number of platelets must be determined twice in each sample, in addition to actually carrying out the test. For this purpose, firstly, special analysis equipment is required and, secondly, every sample must be processed a number of times.
Various commercially available instruments for automated coagulation diagnostics (coagulation analyzers) comprise a centrifugal unit. The latter usually consists of a cuvette rotor, on which a spectrophotometer unit is arranged such that the samples can be measured photometrically during the rotation of the cuvette rotor. It is therefore particularly desirable to provide a method for platelet diagnostics which could be carried out on the available instruments that have a centrifuge unit.